The outcome of hepatitis B virus (HBV) infection results from complicated interplay among a variety of virus-host interactions. Although host responses likely play a major role, this process often depends on a variety of viral adaptive mechanisms. Frequently, viral mutations in critical regions of viral genome are the results of these adaptive mechanisms. Our laboratory has identified and characterized specific viral mutations associated with variant biological behaviors of certain HBV strains. Two mutations in the HBV core promotor were identified in a HBV strain associated with fulminant hepatitis leading to highly enhanced replication as a result of increased viral encapsidation of pregenomic RNA into the core particles. Our recent publications suggest that naturally occurring mutations affecting a novel genetic element may influence viral encapsidation by a co- or post-transcriptional mechanism resulting in enhanced core synthesis and viral replication. As a second aspect of this project, we have initiated studies into the pathogenesis of ground glass hepatocytes in HBV infection. Ground glass hepatocyte is an unique histological feature of chronic hepatitis B virus (HBV) infection. These hepatocytes are stained strongly with anti-HBs and anti-preS1. EM studies show that large amounts of HBV envelope proteins accumulate within dilated vesicles presumably derived from the endoplasmic reticulum. In transgenic mice, overexpression of large surface protein leads to ground glass hepatocytes. The pre-S1 region of the large surface protein has been shown to regulate assembly, processing and secretion of HBsAg. We therefore hypothesize that a mutant form of pre-S1 affects this normal secretory pathway and is responsible for ground glass hepatocytes. To investigate this possibility, we examined HBV sequences spanning the pre-S1 region from HBV infected patients with evident ground glass hepatocytes on liver biopsy. To analyze the viral population of single ground glass hepatocytes, we used the technique of laser capture microdissection to isolate individual hepatocytes from the biopsy specimen. Ground glass hepatocytes that stained positively with anti-HBs as well as normal appearing hepatocytes were harvested individually and their HBV DNA subjected to sequence analysis. Preliminary analysis of one patient revealed that the majority of viral isolates from the ground glass hepatocytes contained a unique pre-S1 mutant sequence. In contrast, the control hepatocytes had exclusively WT sequence. Functional study of this pre-S mutant demonstrated unusual electrophoretic properties and atypical cellular distribution that may have biological implications in the formation of ground glass cells. Additional studies are being pursued in a large number of patients and to analyze the functional effect of these pre-S1 mutations. We have also begun to define and functionally study drug-resistant HBV mutants that were identified in our clinical treatment trial of hepatitis B. Defining the molecular basis of variant manifestations of liver disease associated with infection by naturally occurring or drug-induced HBV mutants may contribute to further understanding of the pathogenesis of HBV infection.